REPLICATION FACTORS AFFECTING GENOME STABILITY:[unreadable] Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase &#948; with 5-flap cutting by FEN1RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3-exonuclease activity of Pol &#948;. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol &#948;) or PIF1 helicase genes can suppress lethality or growth defects of rad27&#916; pol3-D520V mutants (defective for FEN1RAD27 and the 3-exonuclease of Pol &#948;) that produce long flaps, and of dna2&#916; mutants that are defective in cutting long flaps. On the contrary, pol32&#916; or pif1&#916; caused lethality of rad27&#916; exo1&#916; double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short-flap processing activities of FEN1RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery, and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol &#948; is not affected by the presence of Pol32, however, longer flaps only accumulate when Pol32 is present. The presence of FEN1RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol &#948; to FEN1RAD27. Finally, RNA-DNA hybrids are more readily displaced by Pol &#948; than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation. [unreadable] BER[unreadable] [unreadable] DNA double-strand breaks (DSBs) are an important source of genome instability. We are interested in understanding how they are induced, cellular mechanisms for correcting them, and genetic consequences. Among the many ways that they may arise, DSBs can result from closely-opposed breaks in complementary strands that are induced directly by agents such as ionizing radiation. DSBs also might be generated during repair of clustered damage if the repair of closely-opposed lesions were not well-coordinated. To address this possibility we examined the extent to which clustered lesions induced by the alkylating agent methyl methanesulfonate could lead to DSBs in mutants that might affect their repair in the budding yeast Saccharomyces cerevisiae. We used our recently developed approach (Ma et al., 2008), based on detection of DSBs in whole chromosomes by pulsed field electrophoresis analysis, to identify DSBs due to closely-spaced lesions. We found that lack of coordinated polymerase delta and 5'-flap endonuclease Rad27/FEN1 functions during long-patch base excision repair of alkylation damage results in the accumulation of DSBs within the chromosomes of nondividing haploid cells. This was established using mutants combining two defects: i) elimination of the Pol32 subunit of DNA polymerase delta that enhances its processivity via interaction with the PCNA sliding clamp and ii) deletion of the 5'-flap endonuclease Rad27/FEN1, or a mutation that impedes its interaction with PCNA. The DSB accumulation associated with clustered lesions in the double mutants contrasts with the efficient repair of nonclustered lesions, as measured by quantitative PCR and S1 nuclease sensitive sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than widely-spaced damage in order to prevent the development of DSBs.[unreadable] [unreadable] [unreadable] SINGLE-STRAND DNA [unreadable] The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase &#950;. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely-separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.[unreadable] [unreadable] GENE DOSAGE OF GENOME STABILITY GENES [unreadable] We established a system to measure the effect of reduced expression of genome stability genes on chromosome stability in tetraploid yeast, where strains have only 1 copy (simplex) or 4 copies of the gene of interest. Surprisingly, a strain with only one copy of the cohesin gene MCD1 was much more sensistive to ionizing radiation (IR) and methylmethanesulfonate than strains simplex for the RAD50 and RAD51 genes which are central to much of DSB repair. The MCD1 simplex cells, which had been arrested at G2/M, are 10 times more sensitive to the same dose of IR than WT tetraploid with 4 copies. Moreover, total DSB repair was reduced 4 fold in the MCD1 simplex. In contrast, recombination between homologous chromosomes is enhanced about 10-fold for simplex vs 4 copies. These results contrast with UV where there was little, if any, difference with gene copy number. Also, no significant differences were shown between MCD1 simplex and 4 copies cells when G1 stationary cells were irradiated with IR. Based on these findings, DSB repair processes that are determined by recombinational repair mechanisms are dramatically influenced by the dosage of proteins that are identified with maintaining cohesion between sister chromatids